| dc.description.abstract | Fungi are an important group of microorganisms which reproduce by means of specialized cells called spores. These spores are generally considered as a quiescent stage in their life cycle. However, on a suitable substrate, they germinate producing germ tubes which elongate into filaments called hyphae. These hyphae branch, rebranch, and intertwine to form a mass called mycelium. Though the biochemical and genetic aspects of bacterial spore germination have been extensively investigated, such detailed knowledge is lacking in the majority of fungi in general and dermatophytes in particular.
Species of Trichophyton, Epidermophyton, and Microsporum constitute dermatophytes, which cause superficial infection of epidermis, hair, and nails. They reproduce by means of arthrospores, microconidia, and macroconidia. Besides their involvement in the spread of mycotic infections, they provide an ideal experimental system to study morphogenesis and the underlying macromolecular changes (Chapter I).
In the present study, investigations have been carried out with Microsporum canis to understand the RNA metabolism with particular reference to the species of RNA formed. The purification and properties of the DNA-dependent RNA polymerases and in vivo and in vitro transcription have also been studied.
When the morphological changes during the germination of spores of M. canis are followed by light microscopy, germ tubes emerge only after 6 hours. But a significant synthesis of proteins and nucleic acids takes place as revealed by the incorporation of labeled precursors. Rapid uptake of ^32P orthophosphate into the intracellular pools occurs before the synthesis of RNA, which starts around 2 hours and increases up to 6 hours.
The RNA synthesized at the 2nd hour of germination was isolated and analyzed on polyacrylamide gels. The patterns revealed extensive synthesis of rRNA, low molecular weight RNA, and RNA species showing a heterogeneous distribution. In pulse-chase experiments, the label in the heterogeneously distributed RNA was markedly reduced, suggesting the presence of an unstable RNA. By oligo(dT)-cellulose chromatography, this unstable RNA species was confirmed to be mRNA. Similar analysis with 6-hour germinated spores revealed only low levels of mRNA (Chapter II).
Procedures have been standardized to prepare S-30 extracts from mycelia of M. canis, which showed RNA polymerase activity. The activity was resolved into RNA polymerase I, II, and III based on their chromatographic elution on DEAE-cellulose and sensitivity to -amanitin. The three polymerases have been purified to homogeneity by DEAE-Sephadex and affinity chromatography. The properties of these polymerases with respect to their affinity for the native and denatured templates (DNA from M. canis, Micrococcus luteus, salmon sperm, herring sperm) have been studied. They showed increased activity in the presence of manganese as compared to magnesium and a single salt activation profile. Their enzyme kinetics with respect to the effect of substrate concentration have been determined for each of the substrates, viz., ATP, GTP, CTP, and UTP, for all three RNA polymerases. These polymerases have been examined for their subunit structure (Chapter III).
In bacteria, spore-specific genes have been reported. Though the presence of all these RNA polymerases has been reported in a wide range of eukaryotic fungi, the absence of polymerase II in Rhizopus stolonifer suggests the possibility of qualitative and quantitative differences with respect to RNA polymerases from spores and mycelia. Hence, to enable comparison with the corresponding enzymes from mycelia, the RNA polymerases from M. canis spores have been partially purified and characterized. Determination of the activity profiles during early stages of germination has revealed marked increase in RNA polymerase activity up to 12 hours of germination studied. This increased activity may be due to certain proteins regulating transcription by exerting inhibitory or stimulatory effects on RNA polymerases. The proteins from extracts of M. canis mycelia were precipitated with ammonium sulfate (70% saturation) and chromatographed on DEAE-cellulose. The flow-through fraction (unabsorbed basic protein(s)) stimulated mycelial polymerases I and III. These proteins on further fractionation with ammonium sulfate were resolved into stimulatory and inhibitory proteins (Chapter III).
An in vitro analysis of eukaryotic transcriptional system using purified polymerases often fails to provide information on the nature of controls to which RNA polymerases are subjected under in vivo conditions. Among other possibilities, regulation could be effected by the distribution of the polymerase into free and bound states. This aspect could only be examined by studying transcription in isolated intact nuclei. Intact nuclei were isolated from the logarithmically growing cells of M. canis. The free RNA polymerase activity was assayed using poly(dA-dT) as the template in the presence of actinomycin D, which inhibits the bound polymerase. The bound polymerase from log-phase cells showed three-fold higher specific activity as compared to the one from stationary phase. The contribution of polymerase I and II in the bound activity was revealed by their sensitivity to -amanitin (Chapter IV).
The transcription as affected by structural modifications in chromatin could be examined by using an in vitro system having histones, non-histone proteins, and RNA polymerases still bound to the chromatin. One such in vitro system is the "transcriptionally active fraction". It has been isolated both from log-phase and stationary-phase cells of M. canis. The activity in log-phase cells is twice as compared to the stationary-phase cells. The presence of polymerase I and II in the "transcriptionally active fraction" was established by their quantitative differences in the sensitivity to -amanitin. An analysis of in vitro transcripts showed rRNA and 4S RNA, the transcripts of polymerase I and III, respectively.
By oligo(dT)-cellulose chromatography, the presence of poly(A)-containing RNA in the "transcriptionally active fraction" was established. The in vivo transcription using the "transcriptionally active fraction" was visualized by transmission electron microscopy. At the transcriptionally active region, the fanning out of RNP particles was observed, and the active stretches were flanked by supranucleosomal beads. | |
